Caloric restriction without malnutrition improves lifespan in many species. A hallmark of calorie restriction (CR) is insulin sensitivity. The Comprehensive Assessment of the Long-Term Effects of Reducing Intake of Energy (CALARIE) studies have shown that enhanced insulin sensitivity occurs even in non-obese individuals who undergo CR, and it is likely a crucial contributor to various positive health benefits found with CR. In general, across several studies, CR improves systemic peripheral insulin sensitivity by reducing oxidative stress, restoring GLUT4 levels in adipocytes, and thus improving whole-body glucose disposal. However, the molecular mechanism on how this occurs in human is poorly understood. A significant attribute associated with insulin resistance is oxidative stress, which can lead to the production of reactive aldehydes such as 4-hydroxynonenal (4-HNE), that can interact with proteins and alter their function. Emerging evidence suggests that when the protein is GLUT4, a covalent modification (carbonylation) near the glucose transport channel represents just such a factor, which translates to insulin resistance. These studies are the basis of our central hypothesis, which states that the mechanism behind improved insulin sensitivity is reduced carbonyl stress and mitigation of GLUT4 carbonylation. We will test this high risk and high reward hypothesis by determining the stoichiometry of GLUT4 carbonylations in adipose tissue of subjects on sustained CR and compare to a group fed ad libitum. We will then correlate this data to several insulin sensitivity and oxidative stress parameters in the CALARIE database. We also aim to assess the effect of sustained CR on adipose tissue global carbonylome. In summary the proposed studies are designed to address the goals of PA-18-824 which ?is to encourage analyses that will lead to a more detailed understanding of the effects of caloric restriction (CR) on risk factors for chronic diseases, as well as, the cellular/molecular mechanisms mediating the effects of sustained CR in humans.?